Myosin molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in myosins. Examples from this family of molecular motors include myosin II, a non-processive motor that drives cytokinesis and muscle contraction, and myosins V and VI, processive motors that drive vesicular movements in neurons and other cell types. Comparative studies of these three family members have revealed key features of how myosins transduce the chemical energy of ATP hydrolysis into the mechanical energy of movement. The molecular basis of myosin function has been analyzed in detail, following the advent of in vitro motility and laser trap assays, the latter of which allows direct measurement of force and displacement produced by a single myosin molecule pulling on a single actin filament. Data from these assays, among others, provide firm evidence supporting the lever arm hypothesis for the origin of directional movement. Nevertheless, many pivotal issues remain. For instance, we are beginning to understand the importance of tension sensing for modulating the activities and functions of myosin motors. In the coming grant period, we will fully characterize the processive stride of myosin VI, the motor that has most challenged conventional views of myosin structure and function. Using functional analyses of monomeric constructs, we will determine the structural basis of the power stroke and diffusive components of the myosin VI processive stride. We will also determine the molecular basis of tension sensing by myosin V and myosin VI and its significance in processivity. This effort will require our single molecule assays to be further developed using the most current available components. Importantly, we will also develop new technologies to directly visualize the power stroke and nucleotide dynamics of myosins in general to rigorously test current models of myosin movement. PUBLIC HEALTH RELEVANCE All forms of human movement, from the beating of your heart to the processes of individual cell division or cell migration, involve tiny molecular motors that burn the cell's fuel, ATP, in order to produce mechanical force. A wide variety of diseases, including congestive heart failure, cancer, deafness, and a number of neurological syndromes, are the result of defects in these motor proteins. Understanding how they work is therefore fundamental to understanding both their normal and pathologic behaviors.